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Eprints ID : 11087
To link to this article : doi:10.1016/j.funeco.2013.05.007
URL : http://dx.doi.org/10.1016/j.funeco.2013.05.007
To cite this version : Danger, Michael and Chauvet, Eric Elemental
composition and degree of homeostasis of fungi: are aquatic
hyphomycetes more like metazoans, bacteria or plants? (2013) Fungal
Ecology, vol. 6 (n° 5). pp. 453-457. ISSN 1754-5048
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Short Communication
Elemental composition and degree of homeostasis
of fungi: are aquatic hyphomycetes more like
metazoans, bacteria or plants?
Michael DANGERa,*, Eric CHAUVETb,c
a
LIEC e Laboratoire Interdisciplinaire des Environnements Continentaux, University of Lorraine, UMR CNRS 7360,
rue C. Bernard, 57070 Metz, France
b
Universite de Toulouse, UPS, INPT, EcoLab UMR 5245, 118 route de Narbonne, 31062 Toulouse, France
c
CNRS, EcoLab, 31062 Toulouse, France
abstract
Ecological stoichiometry generally assumes that heterotrophs have a higher degree of elemental homeostasis than autotrophs. Differences between fixed consumer nutrient
requirements and nutrients available in resources allow prediction of the intensity of
nutrient recycling ensured by heterotrophs. Despite their fundamental role in detritus
decomposition, extremely few data are currently available on fungal elemental composition.
In this study, we quantified the degree of elemental homeostasis of aquatic hyphomycetes
used as model organisms. Contrary to metazoans, but similar to plants, aquatic hyphomycetes exhibited highly plastic elemental compositions. Mycelium also reached far higher C/
Keywords:
nutrient ratios than reported for bacteria. Our results suggest that non-homeostasis of fungi
Decomposition models
should be explicitly included in stoichiometric models dealing with nutrient recycling, and
Ecological stoichiometry
that the discrepancy in homeostasis between some bacterial strains and fungi should cer-
Elemental composition
tainly be considered when investigating interactions between both groups of decomposers.
Homeostasis
Microbial decomposers
Nutrient recycling
Introduction
Ecological stoichiometry is a recent and powerful theory aimed
at studying the balance of multiple chemical elements in ecological interactions (Sterner and Elser, 2002). Some aspects of this
theory rely on the assumption that heterotrophic organisms
exhibit a higher degree of elemental homeostasis than autotrophic organisms, i.e. a higher resistance to change in their
elemental composition when faced with resources of distinct
elemental composition. For example, several experiments have
* Corresponding author. Tel.: þ33 387378619.
E-mail address: [email protected] (M. Danger).
http://dx.doi.org/10.1016/j.funeco.2013.05.007
shown that an imbalance between a quite constant consumer
demand and variable element supply in resources can predictably affect metazoan’s growth, reproduction and survival (Elser
et al., 1996; Danger et al., 2013a). This imbalance also controls
several ecosystem processes such as nutrient recycling, brought
about by microorganisms (Enrıquez et al., 1993) and metazoans
(Vanni et al., 2002). Currently, most models dealing with microbial nutrient recycling from local to global scales assume fixed
microbial elemental compositions (e.g., Tyrrell, 1999; Daufresne
and Loreau, 2001).
Table 1 e Composition of mineral culture media used for experiments 1 and 2
Compound
Final medium concentration (mg l 1)
Experiments
1 and 2
Experiment 1
NP-rich
MgSO4, 7H2O
CaCl2, 2H2O
FeCl3, 6H2O
MnSO4, H2O
H3BO3
AlSO4, 18H2O
KI
Na2MoO4, 2H2O
CoCl2, H2O
NiCl2, 6H2O
KNO3
Na2HPO4
KH2PO4
N-depleted
P-depleted
Experiment 2
NP-depleted
N/P1
N/P2
N/P3
N/P4
N/P5
100
0.6
0.6
100
1.9
1.9
100
5
5
100
12.5
12.5
100
25
25
0.5
0.15
2
1
1
0.1
0.1
0.1
0.025
0.025
Molar N/P ratio
1 000
50
50
13.8
200
50
50
2.8
While many data on bacteria elemental compositions are
currently available in the literature, data concerning fungi
remain scarce (Persson et al., 2010). Like metazoans, several
bacterial strains are able to maintain their elemental composition at a quite constant level (Chrzanowski and Kyle, 1996;
Makino et al., 2003; Danger et al., 2008). Nevertheless, due to
competitive exclusion mechanisms and selection of bacterial
strains, bacterial communities are likely to adjust their stoichiometry to that of their resources (Danger et al., 2008).
Recent studies also reported a weak homeostasis for some
bacterial taxa depending on resource availability (Scott et al.,
2012). In contrast, data on fungal elemental composition and
on their degree of homeostasis are extremely rare and
incomplete. Yet, such data might be essential for understanding fungal community structures and the intensity of
nutrient recycling. Levi and Cowling (1969) first showed that
some species of fungi were able to reduce their nitrogen (N)
content when facing low N resources. Later, Beever and Burns
(1980) reviewed the phosphorus (P) uptake and storage
capacities of several fungal species showing that some species
were able to store P in excess, but only a few studies were
carried out along nutrient gradients, limiting our abilities to
generalize the lack of homeostasis in fungi. This study was
thus aimed at: (1) testing the degree of elemental homeostasis
of aquatic hyphomycetes, which are generally the most
important fungal decomposers of plant detritus in aquatic
€ rlocher, 1992); and
ecosystems (Suberkropp and Klug, 1976; Ba
(2) giving ranges of variation in fungal mycelium elemental
composition permitting, among others, proper parameterization of models.
Material and methods
Two distinct experiments were carried out. First, we investigated the variation in elemental composition of one aquatic
hyphomycete species, Tetrachaetum elegans, during its growth
in four conditions of nutrient availability (Experiment 1). Four
liquid culture media containing glucose (5 g l 1) as the sole
carbon source and mineral solutions based on the modified
1 000
10
10
68.9
200
10
10
13.8
110
37
13.8
5.5
2.7
GMS medium (Gessner and Chauvet, 1993) were used (Table 1).
The four distinct nutrient levels were achieved by manipulating N and P inputs, leading to a NP-rich treatment,
a N-depleted treatment, a P-depleted treatment, and a NPdepleted treatment. The experiment was carried out in
250 ml Erlenmeyer flasks containing 75 ml of sterilized culture
medium for a total of 72 units, i.e. 4 nutrient levels ! 6
dates ! 3 replicates. Microcosms were agitated on an orbital
shaker and maintained at 15 " C in the dark. Three Erlenmeyer
flasks were randomly sacrificed at each sampling date (2, 4, 6,
8, 11 and 15 d). Mycelium was rinsed twice with sterile
deionized water before being recovered on a pre-weighted GF/
F filter (Whatman International Ltd., Maidstone, UK). Filters
were then frozen at 20 " C before being freeze-dried. Mycelial
biomass was estimated to the nearest 0.1 mg and elemental
composition of mycelium was measured on manually ground
subsamples. Mycelium C and N contents were determined
using a CHN elementary analyzer (NA 1500 Series 2, Fisons,
Manchester, UK) with acetanilide as a standard, and P contents were measured after persulfate digestion in alkaline
conditions followed by colorimetric assessment (Danger et al.,
2008).
In a second experiment (Experiment 2), the degree of
homeostasis of three common aquatic hyphomycete species,
Lemonniera terrestris, Articulospora tetracladia and Tricladium
chaetocladium were assessed using mycelium cultures along a
P-gradient in 45 Erlenmeyer flasks, i.e. 3 species ! 5 Plevels ! 3 replicates. Culture media were similar to those used
in the first experiment (Table 1), except that P inputs were
selected to reach a gradient of molar N/P ratios. This experiment lasted 16 d, i.e. just above the time necessary to reach
steady state for T. elegans in the first experiment. Microcosms
were agitated on an orbital shaker at 15 " C in the dark.
Mycelium analyses were similar to those described for the first
experiment.
For both experiments, flasks were initially inoculated with
1 ml of a suspension of mycelium grown in the NP-depleted
medium. Briefly, single spore isolates of aquatic hyphomycetes were obtained from foam samples taken in headwater
streams of southwestern France. Colonies were grown on 2 %
malt agar. After 15 d, agar plugs containing mycelium were
cut, transferred in sterilized deionized water, and homogenized using a sterile Ultra-Turrax blender (Gessner and
Chauvet, 1993). One ml of each mycelium homogenate was
inoculated into 500 ml-Erlenmeyer flasks containing 150 ml of
sterile NP-depleted medium, and grown for 6 d on an orbital
shaker at 15 " C in the dark. Final mycelium inocula were
obtained from water-rinsed mycelial pellets homogenized in
sterile deionized water with the Ultra-Turrax blender.
In the first experiment, treatment effects on mycelium
biomass, C/N, C/P, and N/P ratios were assessed using twoway ANOVA with nutrient level and time set as fixed factors.
The condition of independence was achieved by destructively
sampling microcosms on each date. For the second experiment, the degree of homeostasis of each species was estimated by quantifying the slopes of the regression between
log(xmedium) and log(xmycelium), x being either C/P or N/P ratios
(Persson et al., 2010). Slopes tending to 1 indicated that
organisms were plastic. Inverse values of the slope (coefficient
of homeostasis H) were also calculated (Sterner and Elser,
2002). All statistical analyses were performed using Statistica
(SAS Institute). The significance threshold was set at P < 0.05.
respectively). These molar ratios were low (9.1 < C/N < 10.2;
66.3 < C/P < 96.4; 7.3 < N/P < 9.4) and typical of organisms with
elevated growth rates (Sterner and Elser, 2002; Makino et al.,
2003). Indeed, high growth rates reduces C/P and N/P ratios of
organisms through increases in their relative requirements of P
compared to other elements due to synthesis of large quantities
of P-rich RNA (heterotrophs: Elser et al., 2003; autotrophs:
!
Agren, 2004; microorganisms: Makino et al., 2003). In addition,
mycelium growing in nutrient unlimited conditions might store
P in excess in their own biomass, mainly in membranes or in
vacuoles (Beever and Burns, 1980). After ca. 8 d of mycelial
growth, nutrient significantly impacted elemental ratios of
fungi (time ! nutrient level: F15, 48 ¼ 21.1, F15, 48 ¼ 68.9, F15,
48 ¼ 53.6, for C/P, C/N, and N/P, respectively; P < 0.001), fungi
tending to match elemental composition of available resources.
At stationary phase, mycelium elemental composition
remained constant and fungal C/nutrient ratios reached far
higher values (up to 1 499 for C/P ratio of L. terrestris) than other
heterotrophic organisms, in particular bacteria. For example,
bacterial C/P ratio never exceeded ca. 500 for both individual
strains and non-homeostatic communities (Chrzanowski and
Kyle, 1996; Makino et al., 2003; Danger et al., 2008; Scott et al.,
2012). These abilities of fungi to live with very limited nutrient
resources certainly explain, in addition to their specific enzymatic activities and production of inhibitory substances, their
competitive advantage in comparison with bacteria on many
nutrient-depleted detrital resources (Gulis and Suberkropp,
2003; Romani et al., 2006; Danger et al., 2013b). Such differences would be representative for organisms with slow (mainly
fungi) versus fast growth (some bacterial strains), as proposed
on stoichiometric basis by Arrigo (2005).
Homeostasis coefficients (1/H and H) of the three different
aquatic hyphomycete strains tested (Table 2) tended to 1,
Results and discussion
Mycelium growth followed similar patterns in all culture media,
i.e. initial exponential growth followed by a stationary phase
(Fig 1A). Mycelial growth did not significantly differ among the
four culture media (nutrient level: F3, 56 ¼ 2.14, P ¼ 0.10;
time ! nutrient level: F18, 56 ¼ 1.25, P ¼ 0.25). At the beginning of
their growth, in nutrient unlimited conditions, mycelium
exhibited similar C/P, C/N, and N/P molar ratios (Fig 1AeC,
500
1250
1000
750
500
400
300
200
100
250
0
0
35
30
C
30
D
25
Mycelial N/P ratio
Mycelial C/N ratio
B
A
1500
Mycelial C/P ratio
Fungal biomass (mg L-1)
1750
25
20
15
10
20
15
10
5
5
0
0
0
2
4
6
8
10
Time (days)
12
14
16
0
2
4
6
8
10
Time (days)
12
14
16
Fig 1 e Growth of the aquatic hyphomycete Tetrachaetum elegans (A) and elemental composition of mycelium (C/P (B), C/N (C),
and N/P (D) molar ratios) during its growth in a batch experiment, in four nutrient conditions: NP-rich ( ), N-depleted ( ),
P-depleted ( ), and NP-depleted ( ). Vertical bars represent standard errors (n [ 3).
Table 2 e Degree of homeostasis of aquatic hyphomycete
species. The coefficient of homeostasis 1/H corresponds
to the slope of the relationship between log(xmedium) and
log(xmycelium), x being the elemental ratios, i.e. C/P or N/P
ratios, as proposed by Persson et al. (2010). All the
regressions were significant (P < 0.001). Slopes and H
values tending to 1 indicate that organisms are plastic
(Persson et al., 2010). Minimal and maximal values of C/P
and N/P molar ratios of mycelium are also displayed
Lemonniera terrestris
Articulospora tetracladia
Tricladium chaetocladium
C/P
N/P
C/P
N/P
C/P
N/P
1/H
H
r2
Range
0.64
0.55
0.75
0.71
0.61
0.68
1.57
1.80
1.34
1.41
1.63
1.47
0.97
0.94
0.98
0.93
0.92
0.93
90e1 499
4.5e52.5
76e1 166
3.6e53.4
89e1 175
3.7e54.8
meaning that these species were highly plastic. For comparison, 1/H for C/P ratios varied between 0.16 and 0.34 for bacterial isolated strains and between 0.07 and 0.20 for
homeostatic zooplankton (Danger et al., 2008; Persson et al.,
2010). Similarly, 1/H for N/P ratios varied between 0.17 and
0.29 for isolated bacterial strains.
To conclude, contrary to what is commonly considered in
most models dealing with nutrient recycling by decomposers
and what has been demonstrated for metazoans (Sterner and
Elser, 2002) and several bacterial strains (Danger et al., 2008;
Makino et al., 2003; but see; Scott et al., 2012), aquatic hyphomycetes are clearly not homeostatic. It is now necessary to
verify the elemental plasticity of other fungi and their physiological determinants in both terrestrial and aquatic contexts
and when facing more refractory C substrates. Models predicting decomposer interactions and nutrient recycling should
explicitly include fungal elemental variability and consider
that overlap can exist between bacterial and fungal elemental
compositions when resources are not nutrient limited, rather
than simply stating that fungi exhibit higher C/nutrient ratios
than bacteria. It would certainly help explain some inconsistencies observed in the outcome of competitive interactions
between fungi and bacteria (e.g., Bengtsson, 1992; MilleLindblom et al., 2006), but also refine predictions of nutrient
recycling models by providing biologically relevant lower and
upper limits of fungal elemental composition.
Acknowledgments
This study was funded through a project of the French
National Program EC2CO ICARE (2012e2013) to MD and the
InBioProcess Project of the ANR Biodiversity Programme
(ANR-06-BDIV-007) to EC. We thank J. Cornut for isolating
 and S. Lamothe for technical assisfungal strains, J. Crampe
€ rlocher for their
tance, and one anonymous reviewer and F. Ba
constructive comments on the manuscript.
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